Interference of KLF9 relieved the development of gestational diabetes mellitus by upregulating DDAH2

Abstract

Gestational diabetes mellitus (GDM) is a situation where glucose intolerance is found in pregnant women without a previous diagnosis of diabetes. The role of Kruppel-like factor 9 (KLF9) has not been investigated in GDM, which constituted the aim of our study. HTR8/SVneo cells were induced by high glucose (HG) and pregnant mice were treated with streptozocin (STZ) to establish GDM model in vitro and in vivo, respectively. The expression level of KLF9 was detected by real-time PCR, immunohistochemical staining, and Western blot. Cell viability, apoptosis, inflammation, and oxidative stress were investigated by cell counting kit-8 (CCK-8), TUNEL, enzyme-linked immunosorbent assay (ELISA) and oxidative stress detection kits, respectively. The interaction of KLF9 with dimethylarginine dimethylaminohydrolase 2 (DDAH2) was predicted by bioinformatic tools and confirmed by luciferase reporter assay and chromatin immunoprecipitation (ChIP). The expression of KLF9 was increased in the placental tissues of GDM patients and HG-induced HTR8/SVneo cells. Silencing of KLF9 increased cell viability, reduced cell apoptosis, and suppressed inflammation and oxidative stress in HG-induced HTR8/SVneo cells. KLF9 could bind to DDAH2 promoter and negatively regulate DDAH2 expression. Inhibition of DDAH2 partly weakened the effects of KLF9 silencing on cell apoptosis, inflammation, and oxidative stress. The suppressive effects of KLF9 silencing on blood glucose and insulin concentration in vivo were also abolished by DDAH2 knockdown. In conclusion, we provided evidence that interference of KLF9 could hinder the development of GDM by alleviating cell apoptosis, inflammation, and oxidative stress through upregulating DDAH2, which might instruct the targeting therapies against GDM.Abbreviations: KLF9: Kruppel-like factor 9; DDAH2: dimethylarginine dimethylaminohydrolase 2 ; GDM: gestational diabetes mellitus; ELISA: enzyme-linked immunosorbent assay; CCK-8: cell counting kit-8; ChIP: chromatin immunoprecipitation; sh: short hairpin; HG: high glucose; PBS: phosphate-buffered saline; DAPI: 4, 6-diamidino-2-phenylindole; IL-6: Interleukin-6; TNF-α: tumor necrosis factor-α; ROS: reactive oxygen species; MDA: malondialdehyde; SOD: superoxide dismutase; wt: wild-type; mut: mutant.

Keywords: DDAH2; KLF9; gestational diabetes mellitus; inflammation.

Figure 1.

KLF9 was high expressed in GDM cells and tissues. (a) The expression of KLF9 in the placental tissues of normal pregnant women and GDM patients. (b) The staining of KLF9 in the placental tissues of GDM patients. Magnification x 100. The (c) protein and (d) mRNA expression of KLF9 in HG-induced HTR8/SVneo cells. ***p < 0.001 vs control.

Figure 2.

Inhibition of KLF9 increased the cell viability and decreased cell apoptosis. After transfection with sh-KLF9-1 or sh-KLF9-2 or sh-NC, the (a) protein expression and (b) mRNA level of KLF9 were detected. ***p < 0.001 vs sh-NC. The (c) cell viability, (d) apoptosis and (e) apoptosis-associated protein levels were measured in HG-induced HTR8/SVneo cells by CCK-8 assay, TUNEL assay and Western blot, respectively. *, ***p < 0.05, 0.001 vs HG; ###p < 0.001 vs HG+sh-NC.

Figure 3.

Inhibition of KLF9 reduced the inflammatory response and oxidative stress in HG-induced HTR8/SVneo cells. (a) The expression of inflammatory cytokines in HG-induced HTR8/SVneo cells transfected with sh-KLF9 was detected by ELISA. (b) The expression of p-p65 and COX-2 in HG-induced HTR8/SVneo cells transfected with sh-KLF9 was detected by Western blot. The detection of (c) ROS, (d) NOS and (e) NO by corresponding kits. *, **, ***p < 0.05, 0.01, 0.001 vs HG; ##, ###p < 0.01, 0.001 vs HG+sh-NC.

Figure 4.

KLF9 transcription inhibits the expression of DDAH2. (a) The binding sites between KLF9 and DDAH2 promoter was predicted by JASPAR. (b) The luciferase activity of the DDAH2 detected by dual luciferase reporter assay. ***p < 0.001 vs sh-NC. (c) The combination between KLF9 and DDAH2 promoter detected by ChIP. ***p < 0.001 vs IgG. The (d) protein and (e) mRNA levels of DDAH2 in HG-induced HTR8/SVneo cells. **, ***p < 0.01, 0.001 vs control. The (f) protein and (g) mRNA levels of DDAH2 in HG-induced HTR8/SVneo cells transfected with sh-KLF9. ***p < 0.001 vs sh-NC.

Figure 5.

DDAH2 is involved in the inhibition of sh-KLF9 on the cell viability of HG-induced HTR8/SVneo cells. The (a) protein expression and (b) mRNA level of DDAH2 after transfection with sh-NC, sh-DDAH2#1, sh-DDAH2#1 was detected. ***p < 0.001 vs sh-NC. HTR8/SVneo cells were induced with HG and transfected with sh-NC/sh-KLF9 or co-transfected with sh-KLF9 and sh-NC/sh-DDAH2. (c)The cell viability was detected by CCK-8 assay. (d) Cell apoptosis was determined using TUNEL assay. (e) apoptosis-associated protein levels were measured by Western blot. ***p < 0.001 vs HG; ###p < 0.001 vs HG+sh-NC; +, ++, +++p < 0.05, 0.01, 0.001 vs HG+sh-KLF9+ sh-NC.

Figure 6.

DDAH2 is involved in the inhibition of sh-KLF9 on the oxidative stress and inflammation of HG-induced HTR8/SVneo cells. The detection of (a) ROS, (b) NOS and (c) NO in HG-induced HTR8/SVneo cells co-transfected with sh-KLF9 and sh-DDAH2 detected by corresponding kits. The (d) inflammatory factors and (e) expression of inflammation-related markers detected respectively by ELISA and Western blot. ***p < 0.001 vs HG; ###p < 0.001 vs HG+sh-NC; +, ++, +++p < 0.05, 0.01, 0.001 vs HG+sh-KLF9+ sh-NC.

Figure 7.

The impact of KLF9/DDAH2 axis on the in vivo GDM mice model. (a) The bood weights, (b) blood glucose and (c) insulin level were measured in GDM mice injected into sh-KLF9 and sh-DDAH2. (d) The oxidative stress indexes and (e) inflammatory cytokines detected in GDM mice using their corresponding commercial kits. ***p < 0.001 vs Control; ###p < 0.001 vs GDM+sh-NC; +++p < 0.001 vs GDM+sh-KLF9.